The goal of this research project is to develop an innovative yet simple therapeutic strategy to eradicate HCV infection early on in HCV infected or HCV/HIV co-infected injecting drug users (IDUs) by engineering lentiviral vectors (LVs) expressing microRNA clusters in the liver. Specifically, we plan to simultaneously inhibit several steps of the HCV life cycle by purposely targeting various segments of the HCV gene and its host cell companion proteins. LVs targeting the liver will be built to deliver three microRNA clusters aiming at: 1. Blockade of HCV docking and entry into hepatocytes by a microRNA cluster targeting the HCV envelope and four host proteins (scavenger receptor B1, CD81, claudin-1 and occludin) involved in HCV entry into hepatocytes 2. Direct suppression of HCV RNA replication by a microRNA cluster targeting various sections (5'UTR, 3'UTR, structural and non-structural regions) of the HCV genome 3. Interferon-mimetic suppression of the HCV life cycle by a microRNA cluster specifically targeting various sections of the HCV genome known to be the targets on interferon-induced microRNAs This cluster attack on the HCV genome may particularly benefit injecting drug users (IDUs) infected with HCV or HIV/HCV because of the more severe course of their diseases, their more limited and often delayed treatment, their poor adherence to current treatments, and higher risk of emergence of resistant HCV strains. Barriers to HCV diagnosis, assessment, treatment, and monitoring as well as considerable treatment- related toxicities result in extremely low treatment uptake and success among IDUs with the currently available therapies. However, improved clinical outcomes occur after successful treatment in patients who achieve a sustained virologic response (SVR). For difficult-to-treat IDUs, we propose to achieve SVR and possible virus eradication by one time systemic delivery to the liver of several miRNAs. This offers the advantage of a simplified yet effective regimen, very few medical interventions, and reduced toxicities and cost. Targeting diferent viral functionalities may dramatically prolong viral suppression and decrease the likelihood of escape as suggested for the HIV-1 virus. Furthermore, such treatment should reduce the significant morbidity and mortality observed in IDUs co- infected with HCV and HIV. ! PUBLIC HEALTH RELEVANCE: There is a pressing need to develop a curative treatment for hepatitis C virus (HCV) infection, an epidemic afflicting 180 million individuals worldwide. Barriers to HCV diagnosis, assessment, treatment, and monitoring as well as considerable treatment-related toxicities result in extremely low treatment uptake and success among injecting drug users (IDUs) infected with HCV or co- infected with HCV and HIV. The goal of this research project is to develop an innovative yet simple therapeutic strategy to eradicate HCV infection early on in HCV or HCV/HIV co-infected IDUs by engineering lentiviral vectors expressing microRNA clusters in the liver.